Polarization dependent surface enhanced Raman scattering system

ABSTRACT

A surface enhanced Raman scattering (SERS) active nanoassembly comprising anisotropically assembled gold nanoparticles in a monolayer double row immobilized on a glass layer is disclosed. The discrete gold nanoparticles are separated by interparticle gaps of 0.5-10 nm that provide hotsites where appropriate excitation creates surface plasmon resonaces and regions of strong and localized electromagnetic fields that enhance Raman signal substantially, 104-1015 fold. An appropriate SERS apparatus comprising the nanoassembly for detecting an analyte is also disclosed. In addition, a method for producing the nanoassembly as well as the application of the nanoassembly or the apparatus comprising the nanoassembly in a method for measuring the SERS signal of an analyte is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of Ser. No. 15/682,847, nowallowed, having a filing date of Aug. 22, 2017 which is a Continuationof Ser. No. 14/921,442, now U.S. Pat. No. 9,772,290, having a filingdate of Oct. 23, 2015.

BACKGROUND OF THE INVENTION Technical Field

The present disclosure relates to an anisotropic surface enhanced Ramanscattering (SERS) nanoassembly of gold nanoparticles. The presentdisclosure further relates to an apparatus comprising the nanoassemblyfor detecting an analyte. Additionally, the present disclosure relatesto a method for producing the nanoassembly as well as its application ina method for measuring the SERS signal of an analyte.

Description of the Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

Surface enhanced Raman scattering (SERS) has become a center of interestin recent science and technology [Y. Kitahama, M. K. Hossain and Y.Ozaki, Raman, Infrared, and Near-infrared Chemical Imaging (Ed: S.Sasic, Y. Ozaki), John Wiley and Sons, Inc., Hoboken, N.J., 2010.; andR. A. Halvorson and P. J. Vikesland, Environ. Sci. Tech., 2010, 44,7749-7755.; and M. K. Hossain and Y. Ozaki, Curr. Sci., 2009, 97,192-201.; and Y. Ozaki, K. Kneipp and R. Aroca, Frontiers ofsurface-enhanced Raman scattering; John Wiley & Sons Ltd., Chichester,2014.; and E. C. Le Ru and P. G. Etchegoin, Principles ofsurface-enhanced Raman spectroscopy and related plasmonic effects,Elsevier, Amsterdam, 2009.; and K. Kneipp, M. Moskovits and H. Kneipp,Surface Enhanced Raman Scattering—Physics and applications, Springer,Heidelberg and Berlin, 2006.; and R. Aroca, Surface-Enhanced VibrationalSpectroscopy, John Wiley & Sons Ltd., Chichester, 2006.—eachincorporated herein by reference in its entirety]. SERS is not onlysimple with single molecule detection capability but also inherits thefine molecular specificity from the Raman effect of the analyte ofinterest [M. K. Hossain, Y. Kitahama, G. Huang, X. Han and Y. Ozaki,Anal. Bioanal. Chem., 2009, 394, 1747-1761.; and S. Schlucker, Angew.Chem. Int. Ed., 2014, 53, 4756-95.; and K. A. Willets, Chem. Soc. Rev.,2014, 43, 3854-3864.; and M. K. Hossain, Mater. Sci. forum, 2013, 754,143-169.; and P. L. Stiles, J. A. Dieringer, N. C. Shah, R. P. VanDuyne,Annu. Rev. Anal. Chem., 2008, 1, 601-626.; and G. Mcnay, D. Eustace, W.E. Smith, K. Faulds and D. Graham, Appl. Specs., 2011, 65, 825-837.; andH. A. Atwater, Sci. Am., 2007, 296, 56-62.; and M. K. Hossain, G. R.Willmott, P. E. Etchegoin, R. Blaikie and J. R. Tallon, Nanoscale, 2013,5, 8945-50.; and P. G. Etchegoin and E. C. Le Ru, Surface Enhanced RamanSpectroscopy (Ed.: S. Schlucker), Wiley-VCH, Weinheim, 2011.; and S. E.J Bell and A. Stewart, Surface Enhanced Raman Spectroscopy (Ed.: S.Schlucker), Wiley-VCH, Weinheim, 2011.; and K. A. Willets and R. P.VanDuyne, Annu. Rev. Phys. Chem., 2007, 58, 267-297.—each incorporatedherein by reference in its entirety]. Since SERS demands the presence ofa metallic nanostructure, the phenomenon results not only fromlight-molecule interactions but also from light-metal interactions. Themain causative factor of dramatic SERS enhancements is now known: “theanalyte” must be at “the hotsite”, which is the region of strong andlocalized electromagnetic (EM) field modulated by the analyte throughits absorption and orientation. Two mechanisms are implicated in theSERS effect: EM and charge transfer (CT). It is widely accepted that theEM mechanism is more important, where surface plasmon resonances (SPRs)are induced at the interface or curvature by incident photons, causingan enormous increase in the EM field. The Raman signal of an analyteunder such conditions will be enhanced by several orders of magnitude,typically 10⁶-10¹⁰ fold. Noble metal nanoparticles, particularly unitdimers with a small interparticle gap, show a sharp plasmon excitationmediated EM field, leading to large signal enhancement, whichfacilitates single molecule detection in SERS [S. Nie and S. R. Emory,Science, 1997, 275, 1102-1106.; and K. Imura, H. Okamoto, M. K. Hossainand M. Kitajima, Nano Lett., 2006, 6, 2173-2176.; and G. Haran, Acc.Chem. Res., 2010, 43, 1135-1143—each incorporated herein by reference inits entirety]. Interestingly, such an EM field enhancement is stronglydependent on the incident polarization, where in-plane polarization tothe interparticle axis induces the strongest enhancement [E. C. Le Ru,M. Meyer, E. Balackie and P. G. Etchegoin, J. Raman Spectros., 2008, 39,1127-1134.; and P. G. Etchegoin, C. Galloway and E. C. Le Ru, Phys.Chem. Chem. Phys., 2006, 8, 2624-2628.; and E. C. Le Ru and P. G.Etchegoin, MRS Bull., 2013, 38, 631-640.; and W. R. C. Somerville, B.Auguie and E. C. Le Ru, J. Quant. Spectros. & Radia. Trans., 2013, 123,153-168.; and E. C. Le Ru, L. Schroeter and P. G. Etchegoin, Anal.Chem., 2012, 84, 5074-5079.—each incorporated herein by reference in itsentirety]. Rigorous theoretical studies as well as some experimentalstudies have been undertaken to verify the mechanism underlying such anenhancement [F. J. Garcia-Vidal and J. B. Pendry, Phys. Rev. Lett.,1996, 77, 1163-1166.; and H. Xu and M. Kall, ChemPhyChem, 2003, 4,1001-1005.—each incorporated herein by reference in its entirety]. Thepolarization dependence of SERS has been investigated using silverdimers, nanorods, and coupled nanowires [M. Suzuki, W. Maekita, Y. Wada,K. Kitajima, K. Kimura, T. Fukuoka and Y. Mori, App. Phys. Lett., 2006,88, 203121-1-203121-3.; and A. R. Tao and P. Yang, J. Phys. Chem. B,2005, 109, 15687-15690.; and A. G. Brolo, E. Arctander and C. J.Addison, J. Phys. Chem. B, 2005, 109, 401-405.—each incorporated hereinby reference in its entirety]. The results of these investigations maythrow light on the fundamental aspects of SERS mechanism(s) as well asanalyte-metal interactions. However, most of the SERS studies have beenperformed using an ensemble system, where hotsites are not isolated butinteract with each other and thus lose their inherent characteristics.

In ensemble SERS measurements, contrary to a single hotsite, manyinterstitials participate in SERS enhancement along with their widevariety of plasmon excitations [M. K. Hossain, Y. Kitahama, V. P. Biju,T. Kaneko, T. Itoh and Y. Ozaki, J. Phys. Chem. C, 2009, 113,11689-11694.—incorporated herein by reference in its entirety]. However,many-particle aggregates or colloid-based nanostructures are reported toprovide isotropic and inhomogeneous SPRs mediated EM field localization[M. K. Hossain, G. G. Huang, T. Kaneko and Y. Ozaki, Chem. Phys. Lett.,2009, 477, 130-134.; and T. Itoh, V. Biju, M. Ishikawa, Y. Kikkawa, K.Hashimoto, A. Ikehata and Y. Ozaki, J. Chem. Phys., 2006, 124,134708-1-134708-6.—each incorporated herein by reference in itsentirety]. Furthermore, a broad SPR excitation peak, which is muchdifferent from the individual narrow excitations of isolated hotsites[L. Novotny and B. Hecht, Principles of Nano-Optics, CambridgeUniversity Press, Cambridge, 2006.—incorporated herein by reference inits entirety]. Hence, limited-particle aggregates or nanostructures witha unique assembly are essential and indispensable to understand SERSenhancement as well as polarization dependent and polarization selectiveSERS characteristics. However, most of the SERS studies have considereda single dimer where the polarization effect was explained, but not theensemble enhancement [Z. Li and H. Xu, J. Quant. Spectros. & Radia.Trans., 2007, 103, 394-401; and K. D. Alexander, M. J. Hampton, S.Zhang, A. Dhawan, H. Xu and R. A. Lopeza, J. Raman Spectrosc., 2009, 40,2171-2175; and D. F. Zhang, Q. Zhang, L. Y. Niu, L. Jiang, P. G. Yin andL. Guo, J. Nanopar. Res., 2011, 13, 3923-3928—each incorporated hereinby reference in its entirety]. Further, fixed polarization has beenadopted for macroscopic samples, and hence, variations in theinterstitials and SPRs have hardly been explored.

In view of the forgoing, one object of the present disclosure is toprovide anisotropic gold nanoassemblies comprising nanoparticles neitherin physical contact nor agglomerated but rather separated by smallinterparticle gaps that provide high SERS activity. Using suchanisotropic gold nanoassemblies allows fine tuning of polarizationdependent and polarization selective SERS measurements and backgroundfluorescence signals with emphasis on spectroscopic measurements withreference to available active sites (i.e. localized EM fields) ratherthan diffraction limited imaging. A further aim of the presentdisclosure is a method and apparatus comprising the SERS active goldnanoassembly for measuring the surface enhanced Raman scattering (SERS)signal of an analyte and/or detecting an analyte. A further aim of thepresent disclosure is to provide a simple one step evaporation assistednanoparticle assembly process to fabricate the anisotropic goldnanoassemblies.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect, the present disclosure relates to a surfaceenhanced Raman scattering (SERS) active nanoassembly comprising i) aglass layer and ii) gold nanoparticles immobilized on the glass layerwherein the gold nanoparticles are anisotropically assembled as amonolayer double row having a long axis.

In one embodiment, the gold nanoparticles have a spherical morphologyand an average diameter of 25-75 nm.

In one embodiment, the gold nanoparticles are anisotropically assembledas a monolayer double row having a long axis of 1-200 μm in length.

In one embodiment, the gold nanoparticles are discrete and separated byinterparticle gaps of 0.5-10 nm.

In one embodiment, the surface enhanced Raman scattering (SERS) activenanoassembly has a SERS enhancement factor of at least 10⁴.

In one embodiment, the surface enhanced Raman scattering (SERS) activenanoassembly is substantially free of surfactants, capping reagentsand/or linkers.

In one embodiment, the surface enhanced Raman scattering (SERS) activenanoassembly further comprises at least one additional SERS active metalselected from the group consisting of silver, copper, platinum,palladium and alloys thereof.

According to a second aspect, the present disclosure relates to anapparatus for detecting an analyte comprising i) the surface enhancedRaman scattering nanoassembly wherein an analyte is adsorbed onto thesurface enhanced Raman scattering nanoassembly ii) a radiation sourceand iii) a detector wherein the radiation source provides incidentradiation on the analyte and the detector is positioned to receivescattered radiation from the analyte and wherein the scattered radiationis used to detect the analyte.

In one embodiment, the apparatus further comprises additional opticalelements to process, focus and/or deflect either the incident radiationfrom the radiation source or the scattered radiation from the analyte.

In one embodiment, the analyte comprises at least one biologicalmolecule selected from the group consisting of a protein, adeoxyribonucleic acid sequence, a ribonucleic acid sequence, an aminoacid, a peptide, a nucleotide, a nucleoside, and a neurotransmitter.

In one embodiment, the analyte comprises at least one dye.

In one embodiment the radiation source is a krypton ion laser providingincident radiation having a wavelength of 400-800 nm or a helium-neongas laser providing incident radiation having a wavelength of 500-650nm.

In one embodiment, the detector is a multichannel charge couple device(CCD).

According to a third aspect, the present disclosure relates to a methodfor measuring the surface enhanced Raman scattering (SERS) signal of ananalyte comprising i) adsorbing an analyte onto the gold nanoparticlesof the the surface enhanced Raman scattering (SERS) active nanoassemblyto form a substrate ii) exciting the substrate with a light source toproduce a Raman signal and iii) detecting and measuring the Raman signalof the substrate wherein the analyte has a Raman signal that is enhancedrelative to the Raman signal of the analyte without the surface enhancedRaman scattering (SERS) active nanoasssembly.

In one embodiment, the analyte has a Raman signal that is enahanced10⁴-10¹⁵ fold relative to the Raman signal of a substantially similaranalyte measured by substantially similar methods without the surfaceenhanced Raman scattering (SERS) active nanoassembly in terms of theRaman signal intensity.

In one embodiment, the light source is polarizable and the Raman signalof the analyte is maximally enhanced when the light source is polarizedalong the plane of the long axis of the gold nanoparticles of thesurface enhanced Raman scattering nanoassembly and wherein the Ramansignal of the analyte is minimally enhanced when the light source ispolarized perpendicular to the plane of the long axis of the goldnanoparticles of the surface enhanced Raman scattering nanoassembly.

In one embodiment, the gold nanoparticles are discrete and separated byinterparticle gaps and the surface enhanced Raman scatteringnanoassembly has maximum electromagnetic near field distributions in theinerparticle gaps in the range of 10-50 dBV/m.

In one embodiment, the light source has a wavelength of 200-1100 nm thatexcites surface plasmons of the surface enhanced Raman scattering (SERS)active nanoassembly.

According to a fourth aspect, the present disclosure relates to a methodfor producing the surface enhanced Raman scattering nanoassemblycomprising i) washing glass slides with an alcohol ii) immersing thewashed glass slides in a suspension comprising gold nanoparticles 25-75nm in diameter and the alcohol, evaporating the excess alcohol to formthe surface enhanced Raman scattering nanoassembly.

In one embodiment, the washed glass slides are immersed at a 15-45°angle releative to the surface of the solution.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is an atomic force microscopy (AFM) image of the prepared surfaceenhanced Raman scattering (SERS) active nanoassembly of 50 nm goldnanoparticles.

FIG. 2A is a scanning electron microscopy (SEM) image of the preparedSERS active nanoassembly.

FIG. 2B is a SEM image of the magnified view of a segment of theprepared SERS active nanoassembly filtered by 100% contrast and 50% lowbrightness to indicate the interparticle gaps between two adjacentnanoparticles.

FIG. 2C is a SEM image of the magnified view of a segment of theprepared SERS active nanoassembly filtered by 100% contrast and 50% lowbrightness to indicate the interparticle gaps between two adjacentnanoparticles.

FIG. 3 is a SERS spectrum of crystal violet (CV) dye adsorbed on theprepared SERS active nanoassembly and a SERS spectrum of crystal violet(CV) dye on a bare glass substrate without the SERS active nanoassembly.

FIG. 4A is a surface plasmon resonance (SPR) image of the prepared SERSactive nanoassembly of 50 nm gold nanoparticles captured by a highresolution charge-coupled detector (CCD) camera.

FIG. 4B is a SERS image of the SERS active nanoassembly of 50 nm goldnanoparticles captured by a high resolution CCD camera of the samesample at the same position as the SPR image of the prepared SERS activenanoassembly of 50 nm gold nanoparticles.

FIG. 5A is a SERS spectra of CV adsorbed onto different segments (#1-#6)of the prepared SERS active nanoassembly.

FIG. 5B is a free hand schematic illustrating the positioning of thedifferent segments (#1-#6) of the prepared SERS active nanoassembly.

FIG. 5C illustrates the SERS intensity of the 1413 cm⁻¹ band of CVobtained at different segments (#1-#6) of the prepared SERS activenanoassembly.

FIG. 6A is a polarization dependent SERS spectra of CV adsorbed on theprepared SERS active nanoassembly obtained at segment #4 with incidentpolarization varying from 0° to 180° with an interval of 30° and thevertical bar representing the photon intensity.

FIG. 6B is a schematic diagram of the polarization dependent SERSexperimental setup.

FIG. 6C is a half polar graph of the C—C bending mode of CV (1413 cm⁻¹)at an interval of 10° of incident polarization.

FIG. 7A is a polarization selective SERS spectra of CV adsorbed on theprepared SERS active nanoassembly obtained at segment #4 with fixedincident polarization while a rotary polarizer is varied from 0° to 180°with an interval of 30°.

FIG. 7B is a schematic diagram of the polarization selective SERSexperimental setup.

FIG. 7C is a half polar graph of the C—C bending mode of CV (1413 cm⁻¹)at an interval of 10° of rotary polarizer.

FIG. 8A is a half polar graph of polarization dependent fluorescenceemission obtained at an interval of 10° of incident polarization.

FIG. 8B is a half polar graph of polarization selective fluorescenceemission obtained at an interval of 10° of rotary polarizer and obtainedat the same position as the half polar graph of polarization dependentfluorescence emission.

FIG. 9A is the mesh, parameters and laser configuration (λ_(exc)=647 nm,D_(AU)=50 nm, d_(gap)=2 nm) adopted. The bar shows the field intensityand one headed arrows demonstrate the respective incident polarizations.

FIG. 9B is an EM near field distribution of gold nanoparticles meshedanisotropically in two rows at 0° polarization relative to the long axisand the corresponding E_(max)=33.61 dBV/m.

FIG. 9C is an EM near field distribution of gold nanoparticles meshedanisotropically in two rows at 30° polarization relative to the longaxis and the corresponding E_(max)=32.36 dBV/m.

FIG. 9D is an EM near field distribution of gold nanoparticles meshedanisotropically in two rows at 60° polarization relative to the longaxis and the corresponding E_(max)=27.61 dBV/m.

FIG. 9E is an EM near field distribution of gold nanoparticles meshedanisotropically in two rows at 90° polarization relative to the longaxis and the corresponding E_(max)=25.44 dBV/m.

FIG. 9F is an EM near field distribution of gold nanoparticles meshedanisotropically in two rows at 120° polarization relative to the longaxis and the corresponding E_(max)=27.61 dBV/m.

FIG. 9G is an EM near field distribution of gold nanoparticles meshedanisotropically in two rows at 150° polarization relative to the longaxis and the corresponding E_(max)=32.36 dBV/m.

FIG. 9H is an EM near field distribution of gold nanoparticles meshedanisotropically in two rows at 180° polarization relative to the longaxis and the corresponding E_(max)=33.61 dBV/m.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Referring now to the drawings, wherein, like reference numeralsdesignate identical or corresponding parts throughout the several views.

According to a first aspect, the present disclosure relates to a surfaceenhance Raman scattering (SERS) active nanoassembly. Surface enhancedRaman spectroscopy or surface enhanced Raman scattering (SERS) refers toa surface sensitive technique that enhances Raman scattering bymolecules adsorbed on rough metal surfaces or by nanostructures.Therefore, one object of the present disclosure is to provide ananoassembly that increases the Raman scattering and signal of adsorbedmolecules before, during, and/or after excitation.

Raman spectroscopy is a spectroscopic technique used to observevibrational, rotational and other low frequency modes in a system. Ramanspectroscopy is commonly used in chemistry to provide a fingerprint bywhich molecules can be identified. It relies on inelastic scattering, orRaman scattering, of monochromatic light, usually from a laser in thevisible, near infrared, or near ultraviolet range. The laser lightinteracts with molecular vibrations, phonons or other excitations in thesystem, resulting in the energy of the laser photons being shifted up orbeing shifted down. The Raman interaction leads to two possibleoutcomes: i) the material absorbs energy and the emitted photon has alower energy than the absorbed photon, known as Stokes Raman scatteringor ii) the material loses energy and the emitted photon has a higherenergy than the absorbed photon, known as anti-Stokes Raman scattering.The energy difference between the absorbed and emitted photoncorresponds to the energy difference between two resonant states of thematerial and is independent of the absolute energy of the photon. Theshift in energy gives information about the vibrational modes in thesystem.

A particular subset of spectroscopy within the realm of Ramanspectroscopy is surface enhanced Raman spectroscopy (SERS). SERS refersto the observation that certain molecules adsorbed on specially preparedmetallic surfaces possess Raman spectrum of greatly increased intensity.Under external radiation an “active site”, “magic site” or “hot site”appears at junctions of or in the vicinity of nanoparticlescorresponding to the phenomenon of localized surface plasmon resonances(LSPRs) mediating intense electromagnetic (EM) field distribution. Thereare two primary theories to the mechanism of the enhancement effect ofSERS. The electromagnetic theory proposes the excitation of localizedsurface plasmons, while the chemical theory proposes the formation ofcharge-transfer complexes. In the electromagnetic theory, the increasein intensity of the Raman signal for adsorbates on particular surfacesoccurs because of an enhancement in the electric field provided by thesurface.

The surface enhanced Raman scattering (SERS) active nanoassembly of thepresent disclosure comprises a glass layer. The nature of this layer isnot viewed as particularly limiting and any suitable material of varyingsize, shape and texture (i.e. smooth, porous, roughened, corrugativeand/or etched) may be envisioned that is non-conductive and providessuitable SERS activity. In a preferred embodiment, the layer is inert,preferably inert such as glass or silicon, preferably glass. The surfaceenhanced Raman scattering (SERS) active nanoassembly further comprisesgold nanoparticles immobilized on the glass layer, wherein the goldnanoparticles are anisotropically assembled as a monolayer double rowhaving a long axis. The glass layer having gold nanoparticlesimmobilized on it in a monolayer double row is referred to herein as the“SERS active nanoassembly”, “active nanoassembly” or “nanoassembly”.

Gold (Au) is a chemical element exhibiting a face centered cubic crystalstructure. In its purest form, it is a bright, slightly reddish yellow,dense, soft, malleable and ductile metal. Chemically, gold is atransition metal and a group 11 element. It is one of the least reactivechemical elements and is solid under standard conditions. The metaloccurs frequently in elemental (native) form, in solid solution serieswith the native element silver and also naturally alloyed with copper,platinum and/or palladium. Less commonly, it occurs in minerals as goldcompounds, often with tellurium. The most common oxidation states ofgold include ⁺1 known as gold (I), Au(I) or aurous compounds and ⁺3known as gold (III), Au(III) or auric compounds. Gold ions are readilyreduced and precipitated as metal.

In a preferred embodiment, the gold nanoparticles of the presentdisclosure substantially comprise elemental gold. The term “goldnanoparticle” as used herein refers to an elemental gold rich material(i.e. greater than 50%, more preferably greater than 60%, morepreferably greater than 70%, more preferably greater than 75%, morepreferably greater than 80%, more preferably greater than 85%, morepreferably greater than 90%, more preferably greater than 95%, mostpreferably greater than 99% elemental gold by weight), which isimmobilized in a monolayer double row assembly onto a glass layer

In addition to elemental gold, various non-elemental gold materialsincluding, but not limited to, gold alloys, metals and non-metals may bepresent in the gold nanoparticle. The total weight of thesenon-elemental gold materials relative to the total weight percentage ofthe gold nanoparticles is typically less than 30%, preferably less than20%, preferably less than 15%, preferably less than 10%, more preferablyless than 5%, more preferably less than 4%, more preferably less than3%, more preferably less than 2%, more preferably less than 1%.

In addition to elemental gold, it is envisaged that the presentdisclosure may be adapted to incorporate gold alloys as the goldnanoparticles. Exemplary gold alloys include, but are not limited to,alloys with copper and silver (colored gold, crown gold, electrum),alloys with rhodium (rhodite), alloys with copper (rose gold, tumbaga),alloys with nickel and palladium (white gold) as well as alloysincluding the addition of platinum, manganese, aluminum, iron, indiumand other appropriate elements or mixtures thereof. In one embodiment,it is envisaged that the present disclosure may be adapted in such amanner that the gold nanoparticles substantially comprise a gold alloy.

In addition to gold, it is envisaged that the present disclosure may beadapted to incorporate at least on additional SERS active metal (capableof surface plasmon resonance under light from 200-1100 nm) selected fromthe group consisting of silver, copper, platinum, palladium and alloysthereof. These metals may be in the form of nanoparticles and may berandomly or non-randomly arranged amongst the gold nanoparticles of thepresent disclosure or in some embodiments fully substituted for the goldnanoparticles of the present disclosure. In a preferred embodiment lessthan 60% of the nanoparticles are an additional SERS active metal,preferably less than 50%, preferably less than 40%, preferably less than30%, preferably less than 25%, preferably less than 20%, preferably lessthan 15%, preferably less than 10%, preferably less than 5% of thenanoparticles are an additional SERS active metal.

Nanoparticles are particles between 1 and 100 nm (10² and 10⁷ atoms) insize. A particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. The exceptionallyhigh surface area to volume ratio of nanoparticles may cause thenanoparticles to exhibit significantly different or even novelproperties from those observed in individual atoms/molecules, fineparticles and/or bulk materials. Nanoparticles may be classifiedaccording to their dimensions. Three-dimensional nanoparticles have alldimensions of less than 100 nm, and generally encompass isodimensionalnanoparticles. Examples of three-dimensional nanoparticles include, butare not limited to, nanoparticles, nanospheres, nanogranules andnanobeads. Two-dimensional nanoparticles have two dimensions of lessthan 100 nm, generally including diameter. Examples of two-dimensionalnanoparticles include, but are not limited to, nanotubes, nanofibers andnanowhiskers. One-dimensional nanoparticles have one dimension of lessthan 100 nm, generally thickness. Examples of one-dimensionalnanoparticles include, but are not limited to, nanosheets,nanoplatelets, nanolaminas and nanoshells. The gold nanoparticles of thepresent disclosure are preferably three-dimensional nanoparticles, butmay also be one-dimensional, two-dimensional, three-dimensional ormixtures thereof.

Nanoparticles are named for the real-world shapes that they appear torepresent. These morphologies sometimes arise spontaneously as an effectof the synthesis or from the innate crystallographic growth patterns ofthe materials themselves. Some of these morphologies may serve apurpose, such as bridging an electrical junction. In a preferredembodiment, the gold nanoparticles of the present disclosure are in theform of a nanoparticle, which is spherical or substantially spherical(e.g. oval, oblong, etc.) in shape. Alternatively, it is envisaged thatthe gold nanoparticles may have a more polygonal shape and may begenerally cubic or rectangular. However, the gold nanoparticlesdisclosed herein may have various shapes other than spheres and may beof any shape that provides desired SERS activity and/or desiredproperties in the resulting nanoassembly. In a preferred embodiment, thegold nanoparticles have a spherical morphology.

In one embodiment, the gold nanoparticles of the present disclosure areenvisaged to be synthesized and formed into a variety of morphologiesincluding, but not limited to, nanoparticles, nanosheets, nanoplatelets,nanocrystals, nanospheres, nanorectangles, nanotriangles, nanopentagons,nanohexagons, nanoprisms, nanodisks, nanocubes, nanowires, nanofibers,nanoribbons, nanorods, nanotubes, nanocylinders, nanogranules,nanowhiskers, nanoflakes, nanofoils, nanopowders, nanoboxes, nanostars,tetrapods, nanobelts, nanaourchins, nanofloweres, etc. and mixturesthereof.

In one embodiment, the gold nanoparticles have uniform shape.Alternatively, the shape may be non-uniform. As used herein, the term“uniform” refers to an average consistent shape that differs by no morethan 10%, by no more than 5%, by no more than 4%, by no more than 3%, byno more than 2%, by no more than 1% of the distribution of goldnanoparticles having a different shape. As used herein, the term“non-uniform” refers to an average consistent shape that differs by morethan 10% of the distribution of gold nanoparticles having a differentshape. In one embodiment, the shape is uniform and at least 90% of thegold nanoparticles are spherical or substantially circular, and lessthan 10% are polygonal or substantially square. In another embodiment,the shape is non-uniform and less than 90% of the gold nanoparticles arespherical or substantially circular, and greater than 10% are polygonalor substantially square.

Nanoparticle characterization may be used to establish understanding andcontrol of nanoparticle and nanoassembly synthesis, assembly andapplication. In one embodiment, it is envisioned that characterizationis done using a variety of techniques. Exemplary techniques include, butare not limited to, electron microscopy (TEM, SEM), atomic forcemicroscopy (AFM), ultraviolet-visible spectroscopy (UV-Vis), dynamiclight scattering (DLS), X-ray photoelectron spectroscopy (XPS), X-rayfluorescence (XRF), powder X-ray diffraction (XRD), energy dispersiveX-ray spectroscopy (EDX), thermogravimetric analysis (TGA), Fouriertransform infrared spectroscopy (FTIR), matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF),Rutherford backscattering spectrometry (RBS), dual polarizationinterferometry and nuclear magnetic resonance or mixtures thereof.

The size of gold nanoparticles may also dictate the level of SERSactivity and enhancement for the nanoassembly described herein. Forspherical or substantially spherical gold nanoparticles, averageparticle size refers to the average longest linear diameter of the goldnanoparticles. For non-spherical gold nanoparticles, such as cubes,squares and/or rectangles the average particle size may refer to thelongest linear dimension and any of the length, width or height. In apreferred embodiment, the gold nanoparticles of the present disclosureare mondispersed with an average particle size of 25-75 nm, preferably30-70 nm, preferably 35-65 nm, preferably 40-60 nm, preferably 45-55 nm,or 50 nm. In another embodiment, the gold nanoparticles of the presentdisclosure are monodispersed with an average particle size of 1-250 nm,preferably 1-200 nm, preferably 1-150 nm, preferably 5-100 nm,preferably 5-75 nm, preferably 10-50 nm. The size may vary from theseranges and still provide an acceptable SERS active nanoassembly.

In a preferred embodiment, the gold nanoparticles of the presentdisclosure are monodisperse, having a coefficient of variation orrelative standard deviation, expressed as a percentage and defined asthe ratio of the particle size standard deviation (σ) to the particlesize mean (μ) multiplied by 100 of less than 25%, preferably less than10%, preferably less than 8%, preferably less than 6%, preferably lessthan 5%, preferably less than 4%, preferably less than 3%, preferablyless than 2%. In a preferred embodiment, the gold nanoparticles of thepresent disclosure are monodisperse having a particle size distributionranging from 80% of the average particle size to 120% of the averageparticle size, preferably 90-110%, preferably 95-105% of the averageparticle size.

In a preferred embodiment, the gold nanoparticles are entirely discretetaken to include monolayer and non-agglomerated. The gold nanoparticlesare discrete and separated by interparticle gaps or an interparticledistance of 0.5-10 nm, preferably 0.75-9 nm, preferably 1.0-8 nm,preferably 1.25-7 nm, preferably 1.5-6 nm, preferably 1.75-5.5 nm, mostpreferably 2.0-5.0 nm. The interparticle distance refers to the shortestdistance between the outer edges of two neighboring gold nanoparticles.The properties of gold nanoparticles may change when particles aggregateand these interparticle gaps provide the hotsites or active sites forSERS enhancement. In a preferred embodiment, the gold nanoparticles ofthe present disclosure have an average surface to surface interparticledistance of less than 200% of their average particle size, preferablyless than 150% of their average particle size, preferably less than 100%of their average particle size, preferably less than 50% of theiraverage particle size, preferably less than 25% of their averageparticle size, preferably less than 10% of their average particle size,preferably less than 5% of their average particle size. For example,spherical gold nanoparticles having a 50 nm diameter may be separated byan average interparticle distance of 2.5-100 nm, preferably 2.5-50 nm,preferably 2.5-25 nm, preferably 2.5-5 nm.

In a preferred embodiment the gold nanoparticles are immobilized on theglass substrate by adsorption defined as the adhesion of atoms, ions ormolecules to a surface creating a film of gold nanoparticles immobilizedon the glass layer. All bonding requirements (be they ionic, covalent,or metallic) of the constituent atoms of the material are filled byother atoms in the material. The exact nature of the bonding depends onthe details of the species involved but the process can generally beclassified physisorption (characteristic of weak van der Waals forces)or chemisorption (characteristic of covalent bonding) or due toelectrostatic attraction. As used herein “immobilized”, “immobilizing”,“adsorbed”, “adsorbing”, “bound” or “binding” refers to the adsorptionand/or chemical binding via strong atomic bonds (e.g. ionic, metallicand covalent bonds) and/or weak bonds such as van der Waals, hydrogen.In a preferred embodiment, the gold nanoparticles are physisorbed ontothe glass layer, leaving the chemical species of both materials intact.In a preferred embodiment, the SERS active nanoassembly is substantiallyfree of surfactants, capping reagents and/or linkers that are often usedto aid the immobilization of SERS active substrates. For SERSapplications, residual surfactants, capping reagents and/or linkers mayprevent Raman analytes from coming close to the center of theinterparticle gaps or hotsites. In addition, surfactants, cappingreagents and/or linkers induce a huge background emission, which isunfavorable for the specific detection of analytes.

Anisotropy is the property of being directionally dependent, as opposedto isotropy, which implies identical properties in all directions. Itcan be defined as a difference, when measured along different axes, in amaterial's physical or mechanical properties, in terms of the presentinvention it refers to the surface enhanced Raman scattering (SERS)activity of the nanoassembly described herein. In a preferredembodiment, the gold nanoparticles of the present disclosure areanisotropically assembled as a monolayer double row having a long axis.In a preferred embodiment the monolayer double row is linear, it isequally envisioned that the monolayer double layer may have curvature.The glass layer of the present disclosure may have a single or aplurality of monolayer double row assemblies of gold nanoparticles andthey may be spaced 100-1000 μm from each other, preferably 250-750 μmfrom each other or 500 μm from each other.

The monolayer double row of the present disclosure comprises a repeating“doublet” of two gold nanoparticles (see FIG. 2A, FIG. 2B and FIG. 2C).In one embodiment, the monolayer double row is substantially free fromanomalies and comprises a uniform monolayer double row of two goldnanoparticles over the entire length of the long axis. As used herein,an “anomaly” is defined as any section of the monolayer double row thatis not a monolayer double row (i.e. a single row, triple row, bilayer,etc. section). In a preferred embodiment, the monolayer double row isgreater than 70% free of anomalies, preferably greater than 75%,preferably greater than 80%, preferably greater than 85%, preferablygreater than 90%, preferably greater than 95% free of anomalies. Inanother embodiment these anomalies occur with low frequency and themonolayer comprises 3-100 doublets without an anomaly, preferably 5-50doublets, preferably 5-25 doublets, preferably 5-10 doublets without ananomaly.

In a preferred embodiment, the long axis of the monolayer double row hasa length of 1-200 μm, preferably 1-150 μm, preferably 1-100 μm,preferably 5-90 μm, preferably 10-80 μm, preferably 15-75 μm, preferably20-70 μm, preferably 30-60 μm, preferably 40-50 μm. In a preferredembodiment, the monolayer double row has a width measured perpendicularto the long axis of 50-200 nm, preferably 60-150 nm, preferably 75-150nm, preferably 85-125 nm, preferably 100-125 nm. In a preferredembodiment, the SERS active nanoassembly of the present disclosure andthe monolayer double row of gold nanoparticles comprise 50-10,000discrete gold nanoparticles, preferably 100-8000, preferably 200-6000,preferably 300-5000, preferably 400-4000, preferably 500-3000 discretegold nanoparticles.

As used herein, the SERS “enhancement factor” refers to the extent thatsurface enhancement increases the intensity of Raman scattering. In apreferred embodiment, the SERS enhancement factor refers to theanalytical enhancement factor (AEF). The AEF can be calculated giventhat C_(RS) and C_(SERS) are the concentrations detected for non-SERSand SERS activity, respectively, I_(RS) is the Raman intensity producedfor a concentration under non-SERS conditions and I_(SERS) is the Ramanintensity for a concentration under SERS substrate activity usingformula (I):

$\begin{matrix}{{AEF} = \frac{C_{RS}I_{SERS}}{C_{SERS}I_{RS}}} & (I)\end{matrix}$

In one embodiment, the surface enhanced Raman scattering (SERS) activenanoassembly of the present disclosure has a SERS enhancement factor ofat least 10⁴, more preferably 10⁴-10¹⁴, more preferably 10⁵-10¹², morepreferably 10⁶-10¹¹, more preferably 10⁷-10¹⁰. In another embodiment,the SERS enhancement factor may refer to the average SERS enhancementfactor or SERS substrate enhancement factor (SSEF) where the SERSintensity is normalized by the number of adsorbed molecules rather thanby the volume concentration in the starting solution. In anotherembodiment, the SERS enhancement factor may refer to the single-moleculeenhancement factor (SMEF).

According to a second aspect, the present disclosure relates to anapparatus for detecting an analyte including the surface enhanced Ramanscattering (SERS) active nanoassembly in any of its embodiments whereinan analyte is adsorbed onto the surface of the SERS active nanoassembly,a radiation source and a detector wherein the radiation source providesincident radiation on the analyte and the detector is position toreceive scattered radiation from the analyte and wherein the scatteredradiation is used to detect the analyte. Any suitable surface enhancedRaman scattering (SERS) apparatus may be used in combination with theSERS active nanoassembly in any of its embodiments of the presentdisclosure. Two exemplary apparatus set ups are shown in FIG. 6B andFIG. 7B.

As used herein, the term “analyte” refers to a chemical or biologicalentity that can be identified, detected and/or quantified by an analyticprocess such as the method of measuring the surface enhanced Ramanscattering (SERS) signal of the analyte described herein. In a preferredembodiment the analyte is a chemical or biological entity that can bedetected or quantified by SERS. A “biological analyte” includes, but isnot limited to, microorganisms, cells, cell products, and/or biologicalmolecules. A microorganism refers to a microscopic living systemincluding, but not limited to, viral particles such as virions, prions,or viriods, bacteria, fungi, archae, protists, microscopic algae,plankton, planarian and mixtures thereof. A cell includes bothprokaryotic and eukaryotic cells, including both natural and recombinantcells and cell products include constituents of cells such as cellmembranes and organelles.

As used herein, “biological molecule” refers to a molecule that isproduced by a living organism and also refers to synthetic analogs ofsuch molecules. Examples of such biological molecules include, but arenot limited to, carbohydrates such as glucose, disaccharides andpolysaccharides, proteins, lipids, lipid bilayers, nucleic acids such asDNA and RNA. Biological molecules may also refer to small molecules,including monomers and oligomers of other biological molecules, i.e.nucleic acids, nucleotides, fatty acids and the like. The biologicalmolecules may be naturally occurring or synthetic and may include bothnaturally occurring and synthetic portions. The term biological moleculealso refers to derivative of biological molecules, such as conjugatednanoparticles. In a preferred embodiment, the analyte comprises at leastone biological molecule selected from the group consisting of a protein,a deoxyribonucleic acid sequence, a ribonucleic acid sequence, an aminoacid, a peptide, a nucleotide, a nucleoside and a neurotransmitter.

A “chemical analyte” refers to an analyte that is not a biologicalanalyte as defined above. In one embodiment, chemical analytes arenon-biological molecules and may be organic, inorganic or a combinationof organic or inorganic moieties. The chemical analyte may be syntheticor naturally occurring, such as a synthetic polymer or synthetic polymernanoparticles. In another embodiment the analyte may comprise a probemolecule referring to biological or chemical analytes that areimmobilized on the nanoassembly and bind or interface with anothercomponent of the analyte. Not limiting examples include, but are notlimited to, antibody-antigen, protein-ligand, protein-aptamer, pairednucleotides and the like.

Dyes which strongly adsorb in the visible light range and are thereforein resonant conditions and are an important family of SERS probes.Acidic dyes are dyes that have a negative charge causing them to bind orassociate with positively charged structures; representative examplesinclude nigrosine, picric acid, eosin, acid fuschin and the like. Basicdyes are dyes that have a positive charge causing them to bind orassociate with negatively charged structures; representative examplesinclude crystal violet, methylene blue, safranin, basic fuschin and thelike. Neutral dyes are generally formed from precipitation in whichaqueous acidic and basic dyes are combined; representative examplesinclude eosinate of methylene blue, giesma and the like. In terms of thepresent disclosure, the analyte may comprise a dye and the dye may beacidic, basic or neutral, preferably basic, preferably crystal violet(CV, C₂₅H₃₀ClN₃).

Crystal violet (also known as gentian violet, methyl violet 10B orhexamethyl paraosaniline chloride) is a triarylmethane dye. Whendissolved in water the dye has a blue-violet color with an absorbancemaximum at 590 nm and an extinction coefficient of 87,000 M-1 cm-1. Thecolor of the dye depends on the acidity of the solution. At a pH of 1.0the dye is green with absorption maxima at 420 nm and 620 nm, while in astrongly acidic solution the dye is yellow with an absorption maximum at420 nm. It is envisioned that other dyes may be used in lieu of crystalviolet. Suitable dyes may be selected from the group including, but notlimited to, methyl violet, fluorescein, prussian blue, egyptian blue,methyl blue, methylene blue, new methylene blue, han purple, potassiumferrocyanide, potassium ferricyanide, methyl violet 6B, methyl violet2B, fuchsine, ararosaniline, ranailine, new fuchsine, magenta II,bromocresol green, malachite green, xanthene dyes (fluorescein, eosine,phloxine, eythrosine, rose bengal), rhodamine dyes (rhodamine 6G),benzotriazole dyes (benzotriazole BTA, benzotriazole dye 2 BTZ),anthraquinone dyes, flavone dyes, arylmethane dyes, protoberberine dyesand mixtures thereof.

In a preferred embodiment, the radiation source is a laser source. Anysuitable radiation source may be used and is envisioned. Exemplary lasersources include, but are not limited to, gas lasers, chemical lasers,excimer lasers, metal vapor lasers, solid state lasers, titaniumsapphire lasers, fiber lasers, photonic crystal lasers, semiconductorlasers, dye lasers, and free electron lasers.

In a preferred embodiment, the radiation source is an ion laser,preferably a krypton or argon laser, more preferably a krypton laser. Anion laser is a gas laser which uses an ionized gas as its lasing medium.An argon laser is one of the families of ion lasers that use a noble gasas the active medium. Argon lasers emit at 13 wavelengths through thevisible, ultraviolet, and near-visible spectrum at 351.1 nm, 363.8 nm,454.6 nm, 457.9 nm, 465.8 nm, 472.7 nm, 476.5 nm, 488.0 nm, 496.5 nm,501.7 nm, 514.5 nm, 528.7 nm and 1092.3 nm, also frequency doubled toprovide 244 nm and 257 nm. If an argon laser is used it is preferablyoperated at 488.0 or 514.5 nm. A krypton laser is an ion laser usingkrypton ions as a gain medium, pumped by electric discharge. Kryptonlasers emit at several wavelengths through the visible spectrum at 406.7nm, 413.1 nm, 415.4 nm, 468.0 nm, 476.2 nm, 482.5 nm, 520.8 nm, 530.9nm, 568.2 nm, 647.1 nm, 676.4 nm, 752.5 nm, 799.3 nm. If a krypton laseris used it is preferably operated at 530.9 or 647.1 nm. Other exemplaryion lasers include, but are not limited to Argon/Krypton lasers,Helium/Cadmium lasers (442 nm and 325 nm), Copper Vapor lasers (578 nmand 510 nm), Xenon lasers, Iodine lasers and Oxygen lasers. In a mostpreferred embodiment, the radiation source is a Kr⁺ laser operating at647.1 nm.

In another embodiment, the radiation source may be a gas laser,preferably a helium-neon laser. A helium-neon laser or HeNe laser is atype of gas laser whose gain medium consists of a mixture of helium andneon (10:1) inside of small bore capillary tube, usually excited by a DCelectrical discharge. The pressure inside the tube is 1 mm of Hg. Themost widely used HeNe laser operates at a wavelength of 632.8 nm in thered part of the visible spectrum. HeNe lasers emit at severalwavelengths at 543.5 nm, 593.9 nm, 611.8 nm, 632.8 nm, 1.1523 μm, 1.52μm, 3.3913 μm. If a HeNe laser is used it is preferably operated at632.8 nm.

In another embodiment, the radiation source may be a laser diode, anelectrically pumped semiconductor laser in which the active laser mediumis formed by a p-n junction of a semiconductor diode. Exemplaryappropriate types of laser diodes include, but are not limited to,double heterostructure lasers, quantum well lasers, quantum cascadelasers, separate confinement heterostructure lasers, distributed Braggreflector lasers, distributed feedback lasers, vertical cavity surfaceemitting lasers (VCSELs), vertical external cavity surface emittinglasers (VECSELs) and external cavity diode lasers. If a laser diodelaser is used it is preferably operated at 785 nm or 830 nm.

In a preferred embodiment, any suitable detector may be used and isenvisioned. Two main categories of optical detectors are used in typicalSERS apparatuses: single channel and multichannel. Single channeldetectors have just one element that accepts light through the exit slitof a monochromator or polychromator. An apparatus containing a singlechannel detector produces a spectrum by rotating the monochromator orpolychromator grating and recording one data point for each gratingposition. In contrast, multichannel detectors collect many data pointssimultaneously without moving a grating or any other part of thespectrograph. This allows much more efficient data collection than forsingle channel detectors, as large amounts of spectral data can becollected in a single exposure. In terms of the present disclosure, thedetector may be single channel or multichannel.

Exemplary single channel detectors include, but are not limited to, asingle photodiode, a photomultiplier (PMT), and an avalanche photodiode(APD). A photodiode is a semiconductor device that converts light intocurrent; the current is generated when photons are adsorbed in thephotodiode. Photodiodes may contain optical filters, built-in lenses andmay have large or small surface areas. A photomultiplier orphotomultiplier tube (PMT) is a photoemissive device in which theabsorption of a photon results in the emission of an electron. Thesedetectors work by amplifying the electrons generated by a photocathodeexposed to a photon flux. PMTs are extremely sensitive detectors oflight in the ultraviolet, visible and near infrared ranges and multiplycurrent in multiple dynode stages. An avalanche photodiode is a highlysensitive semiconductor electronic device that exploits thephotoelectric effect to convert light to electricity that operates athigh speeds and high gain by applying a reverse bias. APDs can bethought of as photodetectors that provide built in first stage of gainthrough avalanche multiplication and are regarded as the semiconductoranalog to photomultipliers. By applying a high reverse bias voltage,APDs show an internal current gain effect due to impact ionization(avalanche effect). In terms of the present disclosure, the detector ofthe apparatus may be single channel and may be single photodiode, aphotomultiplier and/or an avalanche photodiode.

Exemplary multichannel detectors include, but are not limited to, adiode array and a charge coupled device (CCD). A diode array detector ofphotodiode array detector (DAD or PAD) refers to a one-dimensional arrayof hundreds or thousands of photodiodes that can be used as a positionsensor allowing for high speed parallel read out. The DAD has multiplesample side light receiving sections allowing them to obtain informationover a wide range of wavelengths at one time. A charge coupled device(CCD) is a silicon based multichannel array detector of light andbroadly a device for the movement of electrical charge, usually fromwithin the device to an area where charge can be manipulated, forexample conversion into a digital value. This is achieved by shiftingthe signals between stages within the device one at a time. CCDs movecharge between capacitive bins in the device, with the shift allowingfor the transfer of charge between bins. The CCD is a major piece oftechnology in digital imaging. In a CCD image sensor, pixels arerepresented by p-doped MOS capacitors. The capacitors are biased abovethe threshold for inversion when image acquisition begins, allowing theconversion of incoming photons into electron charges at thesemiconductor oxide interface; the CCD is then used to read out thesecharges. The term CCD may also refer to electron multiplying CCDs, CCDcameras, frame transfer CCDs and intensified CCDs. In terms of thepresent disclosure, the detector of the apparatus may be multichanneland may be a diode array and/or a charge coupled device (CCD),preferably a charge coupled device (CCD).

In one embodiment, the apparatus may further comprise additional opticalelements to process the radiation such as optical elements that focusand/or deflect (e.g. lens and/or mirrors) the radiation from theradiation source to provide incident radiation and/or optical elementsthat focus and/or deflect the scattered radiation to provide thescattered radiation to the detector. Exemplary additional opticalelements include, but are not limited to a filter, a reflector, anattenuator, a lens, an optical filter, a notch filter, a holographicnotch filter, an edge filter, a laser rejection filter, imaging optics,focusing optics, collection optics, an objective lens, a mirror, a beamsplitter, a window, a grating, a prism, a laser line rejection device, awavelength selection device, a polychromator, a spectrometer, amonochromator, a half wave plate, a rotary polarizer and mixturesthereof. In another embodiment, the SERS active nanoassembly of theapparatus may be on a stage. The stage may be connected to a controllerwhich may move the stage in linear and/or rotational translationsvertically and/or horizontally as desired, such that different portionsof the nanoassembly may be irradiated by incident radiation and analyzedas desired.

In another embodiment, the apparatus may further comprise additionalimaging elements. Exemplary additional imaging elements may include, butare not limited to a white light (i.e. Xe lamp) dark field condensercombination for observing LSPR images, a CCD camera or a high resolutionCCD camera for observing SPR and SERS imaging as opposed to spectralmeasurements, a variety of monitors and readouts for the images andmixtures thereof. In another embodiment, the apparatus may furthercomprise analytical equipment that may be programmed to process theinformation from the detector and generate a Raman spectrum, this may becontrolled by a computer. In another embodiment, the same or differentanalytical equipment may process the Raman spectrum to identify at leastone analyte, this may be controlled by a computer. The components of theapparatus described herein may all or partially be automated and/orsynchronized with other components of the apparatus.

According to a third aspect, the present disclosure relates to a methodfor measuring the surface enhanced Raman scattering (SERS) signal of ananalyte including adsorbing at least one analyte onto the goldnanoparticles of the SERS active nanoassembly in any of its embodimentsto form a substrate, exciting the substrate with an incident lightsource, to produce a Raman signal, preferably a SERS signal anddetecting and measuring the scattered radiation of the Raman signal ofthe substrate, wherein the analyte has a Raman signal that is enhancedrelative to the Raman signal of the analyte without the SERS activenanoassembly. This method is envisaged to be adapted to be performedusing any suitable SERS apparatus in combination with the SERS activenanoassembly, including the apparatus in any of its embodimentsdescribed herein as an aspect of the present disclosure.

One or more analytes, as previously described, are adsorbed, aspreviously described onto the gold particles of the SERS activenanoassembly. As used herein, a “substrate” refers to the SERS activegold nanoassembly with at least one analyte completely or partiallyadsorbed onto the nanoassembly. In one embodiment, at least one analyteis adsorbed onto at least one gold nanoparticle; the analyte may beadsorbed onto two or more gold nanoparticles. In another embodiment, theanalyte is adsorbed such that at least a portion is within or exposed tothe interparticle gaps or hotsites of the SERS active nanoassembly. Inone embodiment, the analyte may be adsorbed by a dip and wash method ofimmersion.

The substrate is excited by an incident light source, as previouslydescribed. In a preferred embodiment, the incident light or radiationhas a wavelength that excites surface plasmons of the SERS activenanoassembly, specifically the gold nanoparticles of the SERS activenanoassembly and within the substrate. In a preferred embodiment, thesesurface plasmons resonate in the interparticle gaps or hotsites withincident radiation is surface plasmon resonance (SPR). Analyte moleculesin this enhanced EM field are subjected to stronger polarizing effectsand thereby support Raman scattering with higher efficiency. In oneembodiment, the substrate is excited by incident light in a manner suchthat the discrete gold nanoparticles have maximum electromagnetic nearfield distributions in the interparticle gaps in the range of 10-50dBV/m, preferably 15-45 dBV/m, preferably 20-40 dBV/m, preferably 25-35dBV/m, preferably 27-33 dBV/m, preferably 30-33 dBV/m.

For gold substrates, a laser wavelength operating in the range of200-1100 nm allows efficient coupling to surface plasmons within theSERS active nanoassembly, preferably 300-1000 nm, preferably 400-900 nm,preferably 500-800 nm, preferably 500-700 nm, preferably 500-650 nm,preferably 600-650 nm. In a preferred embodiment, the laser exposuretime is less than 5 s, preferably less than 4 s, preferably less than 3s, preferably less than 2.5 s, preferably less than 2 s. In a preferredembodiment, the laser is operated at a power at the substrate surface ofless than 10 mW, preferably less than 8 mW, preferably less than 6 mW,preferably less than 5 mW or 4 mW. These parameters allow for theminimization and avoidance of photodegradation.

In a preferred embodiment, the method of the present disclosure furthercomprises polarization of the incident light by any suitable manner,i.e. a half wave plate. In one embodiment, the incident light may bepolarized at 0-180° relative to the plane of the long axis of the goldnanoparticles, preferably 30-150°, preferably 60-120°, preferably 90°corresponding to polarization along the plane of the long axis of thegold nanoparticles. In a preferred embodiment, the light source ispolarizable and the Raman signal of the analyte is maximally enhancedwhen the light source is polarized along the plane of the long axis ofthe gold nanoparticles of the surface enhanced Raman scatteringnanoassembly of the present disclosure. In a preferred embodiment, thelight source is polarizable and the Raman signal of the analyte isminimally enhanced when the light source is polarized perpendicular tothe plane of the long axis of the gold nanoparticles of the surfaceenhanced Raman scattering nanoassembly of the present disclosure (i.e.0° or 180°).

In another embodiment, the method of the present disclosure furthercomprises polarization of the scattered Raman signal by any suitablemanner, i.e. a rotary polarizer. In one embodiment, the scattered Ramansignal may be polarized 0-180° relative to the plane of the long axis ofthe gold nanoparticles, preferably 0-150°, preferably 0-120°, preferably0-90°, preferably 0-60°, preferably 0-30° relative to the plane of thelong axis of the gold nanoparticles.

With regards to the fluorescence background, the SERS signal loses itsenhancement at a higher background. In a preferred embodiment the lightsource is operated in a manner too weak to induce fluorescence from theanalyte and the background fluorescence is largely due to the surfaceplasmon resonances in the gold nanoassembly. In a preferred embodiment,the light source is polarizable and the surface enhanced fluorescencesignal of the analyte is maximally enhanced when the light source ispolarized along the plane of the long axis of the gold nanoparticles ofthe surface enhanced Raman scattering nanoassembly of the presentdisclosure. In a preferred embodiment, the light source is polarizableand the fluorescence signal of the analyte is minimally enhanced whenthe light source is polarized perpendicular to the plane of the longaxis of the gold nanoparticles of the surface enhanced Raman scatteringnanoassembly of the present disclosure.

The scattered light or scattered Raman signal is detected and measured,as previously described. In a preferred embodiment the Raman signal isdetected in the range of 200-2000 cm⁻¹, preferably 400-1800 cm⁻¹,preferably 600-1600 cm⁻¹, preferably 800-1400 cm⁻¹ depending on theanalyte of interest. In a preferred embodiment the analyte has a Ramansignal that is enhanced (with regards to atomic unit intensity) 10²-10¹⁵fold relative to the Raman signal of a substantially similar analytemeasured by substantially similar method without the surface enhancedRaman scattering (SERS) active nanoassembly (i.e. on a bare glasslayer), preferably, more preferably 10⁴-10¹⁴, more preferably 10⁵-10¹²,more preferably 10⁶-10¹¹, more preferably 10⁷-10¹⁰. This level ofenhancement should be sufficient for single molecule detectionapplications. In another embodiment, the method may further comprisecorrelating the scattered Raman signal from the analyte with a chemicalstructure of a known analyte, this may be done with the use of acomputer.

According to a fourth aspect, the present disclosure relates to a methodfor producing the surface enhanced Raman scattering (SERS) nanoassemblyof the present disclosure in any of its embodiments by wet chemistrytechniques in a simple one step evaporation assisted method. Theassembly of nanoparticles at liquid-liquid, liquid-air and liquid-solidinterfaces is accomplished by the Langmuir-Blodgett technique,sedimentation or evaporation induced self-assembly and the adsorption ofnanoparticles. The Langmuir-Blodgett technique has been used to formnanoparticle monolayers at the water-air interface and to transfer themonto a solid support. Close-packed 2D nanoparticle lattices and 1Darrays with varying surface density of nanoparticles were generated bytuning the wetting and the speed at which the substrate is withdrawn. Inanother embodiment, the hierarchical ordering of nanoparticles at theinterface can be modulated by local heating of the monolayer ofnanoparticles using, for example, irradiation.

Glass slides are washed with an alcohol in an ultrasonic bath for up to2 hours, preferably up to 1 hour, preferably up to 45 min, preferably upto 30 min, preferably up to 20 min, preferably up to 15 min, preferablyup to 10 min, preferably up to 5 min. In terms of the presentdisclosure, suitable alcohol solvents may include, but are not limitedto, the short chain alcohols such as one or more of methanol, ethanol,propanol, isopropanol, butanol of the like. It is envisaged that thecurrent method may be further adapted to use other polar protic solventssuch as water or polar aprotic solvents such as acetone.

The washed glass slides are then immersed in a solution of colloidalgold comprising substantially spherical gold nanoparticles of 25-75 nmin diameter, preferably 30-70 nm, preferably 35-65 nm, preferably 40-60nm, preferably 45-55 nm, or 50 nm in diameter. As used herein, a colloidis a substance in which microscopically dispersed insoluble particlesare suspended throughout another substance.

In one embodiment, the colloidal suspension of gold nanoparticles has amass concentration of less than 5.0 mg/mL, preferably less than 1.0mg/mL, preferably less than 0.75 mg/mL, preferably less than 0.5 mg/mL,preferably less than 0.25 mg/mL, preferably less than 0.2 mg/mL,preferably less than 0.15 mg/mL, preferably less than 0.10 mg/mL,preferably less than 0.05 mg/mL, preferably less than 0.025 mg/mL. Inone embodiment, the colloidal suspension has an atomic gold molarity ofless than 10 mmol/L, preferably less than 5.0 mmol/L, preferably lessthan 2.0 mmol/L, preferably less than 1.0 mmol/L, preferably less than0.75 mmol/L, preferably less than 0.5 mmol/L, preferably less than 0.4mmol/L, preferably less than 0.3 mmol/L, preferably less than 0.2mmol/L, preferably less than 0.1 mmol/L. In one embodiment, thecolloidal suspension of the present disclosure has a particleconcentration of 10⁵-10¹⁵ particles/mL, preferably 10⁶-10¹⁴, preferably10⁷-10¹³, preferably 10⁸-10¹³, preferably 10⁹-10¹³ particles/mL. In oneembodiment, the colloidal suspension has a gold mass percentage byweight of less than 1%, preferably less than 0.5%, preferably less than0.1%, preferably less than 0.05%, preferably less than 0.01%, preferablyless than 0.005%.

In one embodiment, the washed glass slides are immersed in the colloidalgold solution at an inclination of 15-45° relative to the surface of thesolution, preferably 20-40°, preferably 20-35°, preferably 25-30°, or aninclination of 30° relative to the surface of the solution. In oneembodiment, excess ethanol helped in increasing the evaporation rate andpreventing the pile up of nanoparticles at the meniscus. The convectionflow of constituent gold nanoparticles toward the contact area wascontrolled by exposing the meniscus to air. As a result, the contactarea turned into nanostructures in a random fashion. Based on the rateof the ethanol evaporation and transient changes in the localconcentration of gold nanoparticles at the meniscus, different kinds ofnanoassemblies were observed at different vicinities near contact area.The nanoassemblies were all confirmed to be monolayer rather than anagglomeration by atomic force micrscopy (AFM) and scanning electronmicroscopy (SEM).

In another embodiment, it is envisaged that the method to produce thesurface enhanced Raman scattering (SERS) active nanoassembly of thepresent disclosure is not particularly limiting and may be adapted toincorporate a variety of methods that provide highly uniform andreproducible nanoassemblies in any of their embodiments including, butnot limited to, techniques broadly categorized as ion implantation, wetchemistry, physical vapor deposition and mixtures thereof. In oneembodiment, the SERS active nanoassembly may be fabricated by wetchemical methods involving the reduction of a SERS active metal salt(i.e. chloroauric acid or silver nitrate) with a reducing agent (i.e.sodium borohydride) and optionally in the presence of a colloidalstabilizer.

In one embodiment, the nanoassembly may be formed by lithography, morepreferably nanolithography. Nanolithography techniques may becategorized as in serial or parallel, mask or maskless/direct-write,top-down or bottom-up, beam or tip-based, resist-based or resist-lessmethods all of which are acceptable in terms of the present disclosure.Exemplary nanolithography techniques include, but are not limited to,optical lithography, photolithography, directed self-assembly, extremeultraviolet lithography, electron beam lithography, electron beam directwrite lithography, multiple electron beam lithography, nanoimprintlithography, step-and-flash imprint lithography, multiphotonlithography, scanning probe lithography, dip-pen nanolithography,thermochemical nanolithography, therman scanning probe lithography,local oxidation nanolithography, molecular self-assembly, stencillithography, X-ray lithography, laser printing of single nanoparticles,magnetolithography, nanosphere lithography, proton beam writing, chargedparticle lithography, ion projection lithography, electron projectionlithography, neutral particle lithography and mixtures thereof.

The examples below are intended to further illustrate protocols forpreparing and characterizing the surface enhanced Raman scattering(SERS) active nanoassembly of the present disclosure. Further, they areintended to illustrate assessing the properties of these nanoassemblies.They are not intended to limit the scope of the claims.

Example 1

Preparation of the Surface Enhanced Raman Scattering (SERS) ActiveNanoassembly

Colloidal gold nanoparticles (50 nm in diameter) were received fromBBlnternational (Cardiff, UK) and used without further modification. Asimple and modified strategy was adopted to fabricate the anisotropicnanoassembly. Colloidal gold nanoparticles were immobilized on a glasssubstrate without any surfactant or capping reagent. In theself-assembly technique, surfactants or capping reagents act as a meshfor the adhesion of nanoparticles into close packed structure, and thus,the network is converted into a self-assembled nanostructure. However,for SERS applications, residual surfactants may prevent the Raman dyemolecules from coming close to the center of the interstitials. Inaddition, surfactants induce a huge background emission, which isunfavorable for the specific detection of analytes [K. E.Shafer-Peltier, C. L. Haynes, M. R. Glucksberg and R. P. VanDuyne, J.Am. Chem. Soc., 2003, 125, 588-593.—incorporated herein by reference inits entirety].

The gold nanoassembly was prepared in a simple one step process. Glassslides were washed with ethanol in an ultrasonic bath for 15 min andthen immersed at ˜30° inclination in an ethanolic solution of colloidalgold. Excess ethanol helped in increasing the evaporation rate andpreventing the pile up of nanoparticles at the meniscus. The convectionflow of constituent gold nanoparticles toward the contact area wascontrolled by exposing the meniscus to air. As a result, the contactarea turned into nanostructures in a random fashion. Based on the rateof ethanol evaporation and transient changes in the local concentrationof gold nanoparticles at the meniscus, different kinds of nanoassemblieswere observed at different vicinities near the contact area.

Example 2

Surface Enhanced Raman Scattering (SERS) Experimental Setup

The anisotropic nanoassemblies under investigation were found to be SERSactive. Crystal Violet (CV, C₂₅H₃₀Cl N₃, Wako Pure Chemical IndustriesLtd.) molecules were adsorbed to monitor the SERS activity. The preparednanoassemblies were dipped in a 1 μM solution of CV for 15 min andwashed with pure water. It was assumed that only a monolayer of CVremained at the surface, since the interaction between the goldnanoparticles and the CV molecules is stronger than that amongst the CVmolecules.

The SERS measurements were performed using a 647 nm laser from a Kr⁺source [T. Itoh, K. Hashimoto and Y. Ozaki, Appl. Phys. Lett., 2003, 83,2274-2276.—incorporated herein by reference in its entirety]. Theexposure time for SERS imaging was set to 2 s under less than 4 mW oflaser power (density, ˜50.93 W cm⁻²) at the sample surface in order toavoid photodegradation. Surface plasmon resonance (SPR) and SERS imageswere captured at the same spatial position with a slight variation inthe optical path. Hence, it is reasonable to consider that theinterstitials remain unchanged for a particular scattering volume underirradiation.

A high resolution charge-coupled detector (CCD, Hamamatsu photonicsORCA® AG) was attached to the microscope for acquiring SPR and SERSimages. The scattering signal was collected through an objective lens(60×, NA: 0.7) and detected by using a combination of a CCD camera and apolychromator. A half wave plate was inserted in the optical path of thelaser beam to investigate polarization dependent SERS characteristics atthe same position. Between two measurements, the laser was blocked toavoid any unexpected irradiation effect on the sample, although theincident power was kept very low. A rotary polarizer was place in theoptical path of the scattered signal to investigate polarizationselective SERS characteristics at the same position.

Example 3

Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM)Analysis of the Prepared Surface Enhanced Raman Scattering (SERS) ActiveNanoassembly

The nanoassemblies were confirmed to be a monolayer rather than anagglomeration by atomic force microscopy (AFM) and scanning electronmicroscopy (SEM). Atomic force microscopy (AFM) observations confirmedthat the prepared anisotropic gold nanoassembly is several tens ofmicrometers long. A typical AFM image is shown in FIG. 1. A closeobservation by scanning electron microscopy (SEM), as shown in FIG. 2A,confirmed that the constituent gold nanoparticles were neither inphysical contact nor agglomerated, but separated by small interparticlegaps (2-5 nm). As per the hotsite mechanism, such a small interparticlegap is essential for giant SERS enhancement. The EM field is assumed tobe localized and substantially enhanced at such interstitials, and thus,giant enhancement in the SERS is expected.

Example 4

Surface Enhanced Raman Scattering (SERS) and Surface Plasmon Resonance(SPR) Analysis of the Prepared SERS Active Nanoassembly

Indeed, a strong enhancement in the SERS signal of the Crystal Violet(CV, C₂₅H₃₀ClN₃) was observed in the presence of the gold nanoassembly.FIG. 3 shows the Raman signal of CV adsorbed on the gold nanoassemblyand that on a bare glass substrate. The SERS peaks obtained in theseexperiments coincide with those reported by theoretical and experimentalinvestigations [M. V. Canamares, C. Chenal, R. L. Birke and J. R.Lombardi, J. Phys. Chem. C, 2008, 112, 20295-20300.; and S. L. Kleinman,E. Ringe, N. Valley, K. L. Wustholz, E. Phillips, K. A. Scheidt, G. C.Schatz and R. P. VanDuyne, J. Am. Chem. Soc., 2011, 133, 4115-4122.—eachincorporated herein by reference in its entirety]. Clearly, the Ramansignal in the presence of the gold nanoassembly was enhanced by severalorders of magnitude compared to that obtained on the bare substrate. Thenegligible Raman signal observed on the bare substrate could be becauseof sneaking or stray scattering from neighboring nanoparticles. Thescattering volume under investigation was kept at 2 μm×2 μm; therefore,it is likely to have a fraction of stray signals from neighboringSERS-active spots.

Dark-field microscopic measurements were carried out using a combinationof a dark-field condenser and a white light source [J. Nelayah, O.Stephan, M. Kociak, F. J Garcia de Abajo, L. Henrard, I.Pastoriza-Santos, L. M. Liz-Marzán and C. Colliex, Microsc. Microanal.,2007, 13, 144-145.—incorporated herein by reference in its entirety].FIG. 4A shows a dark-field microscopic image of the anisotropic assemblyof 50 nm gold nanoparticles. This image reflects localized surfaceplasmon resonance (SPR) excitations on the substrate. Inhomogeneousintensity distribution was observed from segment to segment of theassembly in the SPR image. The overall topographic observationsconfirmed that the gold nanoparticles remain assembled astwo-dimensional structures rather than as agglomerations, even after theanalyte adsorption. The anisotropic gold nanoassembly was found to betilted ˜20° to the horizontal axis, as indicated in FIG. 4A.

Since SERS is related to the SPR-mediated localized EM field, the samesample was used for far-field Raman measurements in the same platform,with just a slight variation in the optical set up. FIG. 4B shows a SERSimage of the same gold assembly adsorbed with CV as shown in FIG. 4A.The SERS signals were found to be strongly enhanced and inhomogeneouslydistributed along the assembly. It is noted that such a SERS imageessentially corresponds to cumulative SERS photons combined withbackground fluorescence emission. Moreover, Rayleigh leakage orinteractions from the laser itself can interfere with the image [Z. Xie,Y. Lu, H. Wei, J. Yan, P. Wang and H. Ming, Appl. Phys. B, 2009, 95,751-755.—incorporated herein by reference in its entirety]. Hence, thespectral analysis is more reliable than the apparent imaging by thecharge-coupled detector (CCD) camera, as discussed below.

FIG. 5A shows a series of SERS spectra obtained at different positionsmarked 1 to 6, as shown in FIG. 5B. FIG. 5B represents a free handdrawing similar to that of the object undertaken in the presentdisclosure. Inhomogeneous intensity distribution in the SERS image wasobserved from segment to segment. The Raman intensities of the 1413 cm-1SERS band of CV (i.e., the C—C bending mode) obtained at differentsegments are depicted as bar graphs in FIG. 5C. Strong and nearlysimilar signal enhancements are seen at different positions, except atpositions 2 and 4, wherein the signal enhancement is relatively higher(by ˜100 cnts). Ensemble SERS measurements by far-field configurationaverage all the enhanced signals from the total scattering volume (inthis case, 2 μm×2 μm on a planar surface). Hence, the segment withinthis area consists of many sites, with only some of them being active[M. K. Hossain, T. Shimada, M. Kitajima, K. Imura and H. Okamoto,Langmuir, 2008, 24, 9241-9244.—incorporated herein by reference in itsentirety].

Example 5

Polarization Dependent Surface Enhanced Raman Scattering (SERS) Analysisof the Prepared Nanoassembly

In the case of dimers or trimers, a strong SERS signal can be realizedby tuning the laser polarization, whereas in ensemble SERS measurements,the scenario is more complicated. In long range structures, anindividual particle is surrounded by many others, and the local EM fieldis affected accordingly. As a result, polarization dependent andpolarization selective SERS characteristics for a long range specimencannot be explained by the EM mechanism alone [M. Moskovits, Rev. Mod.Phys., 1985, 57, 783-826.—incorporated herein by reference in itsentirety]. Hence, the elongated gold nanoassembly with limitedinterstitials used in the current work is possibly a good candidate forthis purpose.

FIG. 6A shows a series of polarization dependent SERS spectra obtainedat a specific position (position #4 shown in FIG. 5B) with differentincident polarization angles. The positin marked #4 is chosen because ofthe stronger SERS enhancement. The experimental configuration isschematically depicted in FIG. 6B, where the incident laser is polarizedusing a half wave plate and the scattering signal is collected by theobjective lens positioned behind the stage; thus, the substrate is leftuntouched for the entire series of experiments. FIG. 6C represents thehalf polar graph for the 1413 cm-1 SERS band of CV (i.e., the C—Cbending mode) obtained at an interval of 10° of the incidentpolarization.

In polarization dependent measurements, the peak intensity changedgradually and reached the highest value at an angle of 70°. From thetopographic measurements, it was found that the anisotropic nanoassemblyis tilted by ˜20° from the horizontal axis. Hence at 70° polarization,the incident laser beam coincides with the long axis of thenanoassembly. A plausible reason for this is described below withreference to the localized EM field distribution at various incidentpolarizations.

In simple nanoparticle dimers, giant enhancement in SERS can be obtaineddue to localized EM field at interstitials that are known as “hotsites”.When the proximity between two nanoparticles becomes very close, theoptical field at interstitials reaches its highest. The phenomenon istermed the “EM enhancement factor” which is usually considered to beproportional to the fourth power of the ratio between the local electricfield and the incident field. Extensive theoretical studies have beenreported [P. K. Aravind and H. Metiu, J. Phys. Chem., 1982, 86,5076-5084.; and J. P. Kottmann, O. J. F. Martin, D. R. Smith and S.Schultz, Phys. Rev. B, 2001, 64, 235402-1-235402-10.—each incorporatedherein by reference in its entirety]. On the other hand, for anaggregate of many nanoparticles, the hotsites become unpredicted andusually occur due to symmetry breaking [K. Kneipp, Y. Wang, H. Kneipp,I. Itzkan, R. R. Dosari and M. S. Feld, Phys. Rev. Lett., 1996, 76,2444-2447.˜incorporated herein by reference in its entirety]. Inaddition, it has been reported that neighboring hotsites always tend tohave coalescence and hybridization through intense energy percolation[M. K. Hossain, T. Shimada, M. Kitajima, K. Imura and H. Okamoto, J.Micros., 2008, 229, 327-330.—incorporated herein by reference in itsentirety].

This was the main motivation to choose a suitable specimen that lay inbetween single dimer and large nanoaggregates. The nanoassembly underinvestigation was an interstitials limited SERS active substrate. Insuch an assembly, a single hotsite occurring at polarization across thelong axis was expected to be undisturbed in an ideal case, whereas allthe interstitials along the long axis will be active at in planepolarization. A theoretical validation was also carried out and isexplained below.

Example 6

Polarization Selective Surface Enhanced Raman Scattering (SERS) Analysisof the Prepared Nanoassembly

It is well established that the SERS enhancement depends on incidentpolarization direction. Stronger SERS enhancement appears for parallelpolarization with reference to the interparticle axis and vice versa.However, the polarization selective SERS signal is not so straightforward, because of unknown adsorption orientation of molecules andnanoscale topography of the underlying substrate. According to the twofold EM enhancement mechanism, the scattering signal interacts with thelocalized plasmons and is modulated. Since a localized EM field isincident polarization specific, polarization selective SERScharacteristics should reflect the scattering signal and SPRsexcitation. A uniform enhancement in polarization selective SERSmeasurements was observed.

FIG. 7A shows a series of polarization selective SERS spectra recordedat the same position (position #4 showed in FIG. 5B). The position #4 ischosen because of the stronger SERS enhancement and for comparison withpolarization dependent SERS. The experimental configuration isschematically depicted in FIG. 7B, where the incident laser polarizationis unchanged and the scattering signal is passed through the rotarypolarizer and collected by the objective lens before the spectrometer.FIG. 7C represents the half polar graph for the 1413 cm⁻¹ band of CVobtained at an interval of 10° of polarization. It is noteworthy thatthe polarization selective peak intensity remains more or less unchangedat a particular incident polarization.

Example 7

Fluorescence Analysis of the Prepared Surface Enhanced Raman Scattering(SERS) Active Nanoassembly

Regarding the fluorescence background, the SERS signal loses itsenhancement at a higher background and vice versa [M. K. Hossain, G. G.Huang, T. Kaneko and Y. Ozaki, Phys. Chem. Chem. Phys., 2009, 11,7484-7490.—incorporated herein by reference in its entirety]. Twofactors must be considered for fluorescence background emission at aspecific adsorption of an analyte to the substrate: i) the excitationlaser itself and ii) the SPR excitation from the underlying substrate.The excitation wavelength of 647.2 nm from the Kr⁺ laser is too weak toinduce fluorescence from CV. Hence, the background emission is mainlydue to SPR excitation in the gold nanoassembly. The SPR image shown inFIG. 4A represents an inhomogeneous distribution of SPR excitationsalong the assembly. It has been shown that several SPR excitations arepossible in elongated and long range colloidal gold nanostructures [M.K. Hossain, Y. Kitahama, G. G. Huang, T. Kaneko and Y. Ozaki, Appl.Phys. B, 2008, 93, 165-170.—incorporated herein by reference in itsentirety]. At position #4, weak fluorescence background emissions wererecorded, as mentioned previously. Although the excitation intensity istoo weak to induce fluorescence in this case, incident polarizationdependent fluorescence emissions were observed for the elongated goldnanoassembly. In fact, fluorescence emission enhancement resulted fromthe SPR mediated local EM field at the interstitials.

FIG. 8A presents the half polar graph of fluorescence emission obtainedat an interval of 10° incident polarization. It is noteworthy that theintensity reaches the highest value at an angle of 70°, similar to thecase of polarization dependent SERS. Since the anisotropic assembly istilted by ˜20°, at 70° polarization, the incident laser beam coincideswith the long axis of the nanoassembly. On the other hand, uniformfluorescence emission on average was observed in the polarizationselective measurements, as shown in FIG. 8B. According to surfaceenhanced fluorescence (SEF), the fluorescence intensity depends on theposition of the molecule from the surface and the intensity of thelocalized EM field mediated by SPR. In the case of a dry sample, anyvariation in the molecule's position is discarded, and localized EMfield distribution can be manipulated by incident polarization. Hence,similar to SERS, fluorescence emission is enhanced at a stronger EMfield and vice versa. It is well understood that in-plane polarizationto the interparticle axis introduces the highest localized EM field atthe interstitial. On the other hand, in the case of polarizationselective SERS measurements, the incident polarization is kept unchangedand the scattering signal is filtered by a rotary polarizer. Since thereis no variation in the localized EM field distribution (because of fixedincident polarization), the fluorescence background emission remainsapproximately uniform, as observed in FIG. 8B. The observations can bedemonstrated through the two fold EM field enhancement mechanism.

Example 8

Three-Dimensional (3D) Finite-Difference Time-Domain (FDTD) Analysis

The giant SERS enhancement, which is typically on the order of 10⁸-10¹⁰,has been mentioned earlier and can be explained by a two-fold EM fieldenhancement mechanism [B. Pettinger, J. Chem. Phys., 1986, 85,7442-7451.; and T. Itoh, K. Yoshida, V. Biju, Y. Kikkawa, M. Ishikawaand Y. Ozaki, Phys. Rev. B, 2007, 76, 085405-1-085405-5.—eachincorporated herein by reference in its entirety]. A quantum mechanicalapproach for surface enhanced optical processes summarizing two-foldenhancements was first reported where two main interactions areresponsible: i) incident photon SPR interaction and scattered photon SPRinteraction. Since the incident photon is much stronger than thescattered one, the first interaction is predominant. In other words, theincident photon energy irradiated on the adsorbent is scattered from anadsorbate because of SPR mediated dipole-dipole interactions. Inaddition, the scattered photon from the molecules resonates with theplasmon, as its wavelength is close to that of the incident photon. TheEM field is further enhanced and thus, cumulative strong Ramanscattering light is emitted. Hence, the total enhancement factor, M, isgiven by formula (II):

$\begin{matrix}{M = {{{\frac{E^{LOC}\left( \lambda_{L} \right)}{E^{I}\left( \lambda_{L} \right)}}^{2} \times {\frac{E^{LOC}\left( {\lambda_{L} \pm \lambda_{R}} \right)}{E^{I}\left( {\lambda_{L} \pm \lambda_{R}} \right)}}^{2}} = {{M_{1}\left( \lambda_{L} \right)} \times {M_{2}\left( {\lambda_{L} \pm \lambda_{R}} \right)}}}} & ({II})\end{matrix}$

In formula (I), M is the total enhancement factor and E^(I) and E^(Loc)are the amplitudes of the incident and local electronic fields,respectively; λ_(L) is the excitation wavelength; +λ_(R) and −λ_(R) arethe wavelengths of the anti-Stokes and Stokes Raman shifts,respectively; and M₁ and M₂ are the first and second enhancementfactors, respectively.

As noted above, the EM distribution at the interstitial sites stronglydepends on the incident polarization. The highest confinement isobserved when the incident polarization is parallel to the interparticleaxis, and thus, the SERS and SEF enhancement under these conditions isthe highest. On the contrary, the lowest enhancement is observed withthe incident polarization normal to the interparticle axis. Athree-dimensional (3D) FDTD analysis, with the same parameters usedunder the experimental conditions (λ_(exc)=647 nm, D_(Au)=50 nm, Gap=2nm), was carried out to understand the observations.

The incident laser polarization was tuned to observe the EM fielddistribution, since the SERS and SEF enhancement in this particular casewas found to be dependent on the incident laser polarization. For thetheoretical calculations to be relevant to the experimentalmeasurements, eight nano-objects were inserted into the mesh in tworows, so that collective EM field distribution could be realized. Insuch a configuration, 10 interstitials are obtained, 6 sites aresupposed to be the strongest at in-plane polarization to the long axisand the remaining 4 sites are the strongest at out-of-planepolarization. The mesh, parameters, and laser configuration are shown inFIG. 9A. FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G, and FIG.9H show the near-field distribution from 0° to 180° incidentpolarization, at intervals of 30°. The intensity bar in FIG. 9Brepresents the intensity scale for all images.

For in-plane polarization to the long interparticle axis (i.e., 0° and180° polarization), the strongest EM near-field distributions, withE_(max)=33.61 dBV/m, were found to occur at the six interstitials alongthe the horizontal axis (FIG. 7B and FIG. 7H). The EM near-fielddistribution for the other sites along the vertical axis was negligible.In such a scenario, the SERS and SEF signals from the analyte ofinterest should show the highest enhancement. For orthogonalpolarization (90° polarization), the weakest EM near-fielddistributions, with E_(max)=25.44 dBV/m, were observed at the fourinterstitials along the vertical axis, as shown in FIG. 7E. For 30°(FIG. 7C and FIG. 7G) and 60° (FIG. 7D and FIG. 7E) polarization, the EMnear-field intensity was 32.36 dBV/m and 27.61 dBV/m, respectively.Hence, in the case of orthogonal polarization, the SERS and SEF signalsfrom the analyte of interest should show minimum enhancement.

Indeed, a similar trend was observed in this investigation. As explainedin FIG. 6C and FIG. 8A, a strong SERS signal for the 1413 cm-1 band ofCV and fluorescence background emission were recorded at 70° incidentpolarization. Since the gold nanoassembly is tilted by ˜20° from thehorizontal axis, 70° polarization of the incident laser becomes in-planepolarization to the long axis. In the case of in-plane polarization tothe long axis, ensemble EM near-field averages the maximum number ofhotsites, and consequently, the SERS and SEF intensities are enhanced.On the other hand, in the case of orthogonal polarization (160° and 20°polarization, as shown in FIG. 6C and FIG. 8A), the gold nanoassemblyinduced low enhancement in the SERS and SEF emission.

Thus, the foregoing discussion discloses and describes merely exemplaryembodiments of the present invention. As will be understood by thoseskilled in the art, the present invention may be embodied in otherspecific forms without departing from the spirit or essentialcharacteristics thereof. Accordingly, the disclosure of the presentinvention is intended to be illustrative, but not limiting of the scopeof the invention, as well as other claims. The disclosure, including anyreadily discernible variants of the teachings herein, defines, in part,the scope of the foregoing claim terminology such that no inventivesubject matter is dedicated to the public.

The invention claimed is:
 1. A polarization dependent surface enhancedRaman scattering system, comprising: a surface enhanced Raman scattering(SERS) active nanoassembly, comprising: a glass layer; and goldnanoparticles immobilized on the glass layer; wherein the goldnanoparticles are anisotropically assembled as a monolayer double rowhaving a long axis, and wherein the gold nanoparticles have a sphericalmorphology, wherein an analyte is adsorbed onto the surface enhancedRaman scattering (SERS) active nanoassembly; a laser radiation source;an objective lens; a wave plate; and a detector; wherein the laserradiation source is disposed above the wave plate which is disposedabove the SERS active nanoassembly which is disposed above the objectivelens which is disposed above the detector, wherein the laser radiationsource provides incident radiation on the analyte and the detector ispositioned to receive scattered radiation from the analyte; and whereinthe analyte is detected by the scattered radiation.
 2. The polarizationdependent surface enhanced Raman scattering system of claim 1, whereinthe laser radiation source is a krypton ion laser that provides incidentradiation having a wavelength of 400-800 nm or a helium-neon gas laserthat provides incident radiation having a wavelength of 500-650 nm. 3.The polarization dependent surface enhanced Raman scattering system ofclaim 1, wherein the detector is a multichannel charge coupled device(CCD).